Dataset Information

Effects of ROCK Inhibitors on Apoptosis of Corneal Endothelial Cells in CMV-Positive Posner-Schlossman Syndrome Patients.

ABSTRACT: Purpose:To examine the role of aqueous tumor necrosis factor ? (TNF-?)-RhoA-Rho kinase (ROCK) signaling in cytomegalovirus (CMV)-induced apoptosis and the barrier function of cultured human corneal endothelial cells (hCECs) in CMV-positive Posner-Schlossman syndrome (CMV+/PSS) patients. Methods:Aqueous levels of TNF-?, IL-8, IL-10, and several other cytokines in 19 CMV+/PSS patients and 20 healthy control subjects were quantitated using a multiplex assay. The expression of active RhoA in hCECs post-CMV infection was determined using western blotting (WB). The expression levels of TNF-? and nuclear factor kappa B (NF-?B) in CMV-infected hCECs were examined by immunocytochemistry (ICC) and WB with and without ROCK inhibitors. The apoptotic rate and barrier integrity in CMV-infected hCECs were also examined. Results:The expression levels of TNF-?, monocyte chemoattractant protein-1 (MCP-1), IL-8, and IL-10 were upregulated in the aqueous humor of CMV+/PSS patients, and among these upregulated cytokines aqueous TNF-? was negatively correlated with the number of corneal endothelial cells. In CMV-infected hCECs, upregulation of TNF-? and NF-?B was determined by WB and ICC. In hCECs, CMV infection induced apoptosis and significantly impaired cell-cell contacts, effects that were attenuated by treatment with a ROCK inhibitor. Conclusions:Aqueous TNF-? was upregulated in CMV+/PSS patients, which may have triggered corneal endothelial cell loss. Modulation of TNF-?, including its downstream Rho-ROCK signaling, could serve as a novel treatment modality for corneal endothelial cell loss in CMV+/PSS patients.

SUBMITTER: Igarashi N

PROVIDER: S-EPMC7441372 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Effects of ROCK Inhibitors on Apoptosis of Corneal Endothelial Cells in CMV-Positive Posner-Schlossman Syndrome Patients.

Igarashi Nozomi N Honjo Megumi M Kaburaki Toshikatsu T Aihara Makoto M

Investigative ophthalmology & visual science 20200801 10

<h4>Purpose</h4>To examine the role of aqueous tumor necrosis factor α (TNF-α)-RhoA-Rho kinase (ROCK) signaling in cytomegalovirus (CMV)-induced apoptosis and the barrier function of cultured human corneal endothelial cells (hCECs) in CMV-positive Posner-Schlossman syndrome (CMV+/PSS) patients.<h4>Methods</h4>Aqueous levels of TNF-α, IL-8, IL-10, and several other cytokines in 19 CMV+/PSS patients and 20 healthy control subjects were quantitated using a multiplex assay. The expression of active ...[more]

PMID: 32749463

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Project description:Objective: Posner-Schlossman syndrome (PSS), also known as glaucomatocyclitic crisis, is an ocular condition characterized by recurrent attacks of anterior uveitis and raised intraocular pressure. Previous studies by our team and others have identified the genetic association of complement pathway genes with uveitis and glaucoma. This study aimed to investigate the complement genes in PSS patients with the view of elucidating the genetic background of the disease. Methods: A total of 331 subjects (56 PSS patients and 275 controls) were recruited for this study. We selected 27 variants in six complement pathway genes (SERPING1, C2, CFB, CFH, C3, and C5) and detected them using TaqMan single nucleotide polymorphism (SNP) Genotyping Assays. Univariate SNP association analysis, haplotype-based association analysis, gene-gene interaction analysis among complement genes, and genotype-phenotype correlation analysis were performed. Results: Among the 27 variants of six complement pathway genes, the functional variant I62V (rs800292) at the CFH gene was found to be significantly associated with PSS; there was a significant increase in the frequency of A allele and AA homozygosity in PSS patients than in controls (P = 1.79 × 10-4; odds ratio (OR) 2.18, 95% CI: 1.44-3.29; P = 4.65 × 10-4; OR 3.66, 95% CI: 1.70-7.85, respectively). The additive effect of CFH-rs800292 and SERPING1-rs3824988 was identified with an OR of 12.50 (95% CI: 2.16-72.28). Genotype-phenotype analysis indicated that the rs800292 AA genotype was associated with a higher intraocular pressure and higher frequency of recurrence. Unlike a high proportion of human leukocyte antigen (HLA)-B27 positivity in anterior uveitis, only 3 in 56 (5.36%) PSS patients were HLA-B27 positive. In addition, one haplotype block (GC) in the SERPING1 gene showed a nominal association with PSS with an increased risk of 2.04 (P = 0.01; 95% CI: 1.18-3.53), but the P-value could not withstand the Bonferroni correction (P corr > 0.05). Conclusion: This study revealed a genetic association of a CFH variant with PSS as well as its clinical parameters, implying that the alternative complement pathway might play an important role in the pathogenesis of PSS. Further studies to enrich the understanding of the genetic background of PSS and the role of the complement system in ocular inflammation are warranted.

Project description:Posner-Schlossman-Syndrome (PSS) is clinically characterized by acute, recurrent, mild, unilateral uveitis anterior accompanied by elevated intraocular pressure (IOP). Fuchs´ Uveitis (FU) is a chronic, low-grade-inflammatory disorder, involving anterior uvea and vitreous. The clinical findings show remarkable similarities as well as differences. In our study, we determine the composition of immune mediators in aqueous humor of patients with PSS and FU and evaluate if immune mediators play a crucial role in specific viral intraocular inflammation and IOP rises. Aqueous humor samples from 81 uveitis patients (= eyes) presenting with either PSS or FU were collected at one time point. Local intraocular antibody synthesis to rubella virus was confirmed in 65 patients, whereas 16 were tested positively for human cytomegalovirus. Thirteen patients with PSS and 10 patients with FU were treated with glaucoma medications. Additionally, 11 cataract patients acted as control group. Immune mediator concentrations were measured by Bio-Plex Pro assay. We observed in both PSS (IFN-γ: 174.9 pg/mL; TNF-α: 25.1 pg/mL) and FU (IFN-γ: 25.4 pg/mL; TNF-α: 27.2 pg/mL) groups a significantly increased level of T-helper 1 immune mediators compared to controls (IFN-γ, TNF-α: 0 pg/mL) [median]. Notably, PSS patients (IL-1RA: 73.4 pg/mL; IL-8: 199.4 pg/mL; IL-10: 33.4 pg/mL; IP-10: 126350 pg/mL) showed a stronger and more active ocular inflammatory response, than FU patients (IL-1RA: 4.3 pg/mL; IL-8: 72.4 pg/mL; IL-10: 1.6 pg/mL; IP-10: 57400 pg/mL). Furthermore, a negative correlation between mediators and IOP was seen in the PSS group, potentially caused by acetazolamide-treatment. Our findings show that immune mediators play a crucial role in specific viral intraocular inflammation and influence IOP levels. Remarkable similarities but also significant differences of immune mediator concentrations are apparent in PSS compared to FU. High concentrations of IL-1RA, IL-8, IL-10, and IP-10 correlate with active inflammation in PSS, while FU may trigger chronic inflammation. Our data also substantiated a very similar composition of cytokines in those patients from the PSS group suffering from ocular hypertension and thus offers a potential explanation model for a negative correlation between mediators and IOP.

Project description:The Posner-Schlossman syndrome (PSS) is a disease with clinically recurrent unilateral anterior uveitis with markedly elevated intraocular pressure (IOP) and subsequent progression to optic neuropathy. Retrospective studies have reported increased annual incidence of PSS, especially in China. While currently, the clinical management of PSS is still challenging. Metabolomics is considered to be a sensitive approach for the development of novel targeted therapeutics because of its direct elucidation of pathophysiological mechanisms. Therefore, we adopted gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) technology-based non-targeted metabolomics approach to measure comprehensive metabolic profiles of aqueous humor (AH) samples obtained from patients with PSS, with an aim to demonstrate the underlying pathophysiology, identify potential biomarkers specific to PSS, and develop effective treatment strategies. A comparative analysis was used to indicate the distinct metabolites of PSS. Pathway analysis was conducted using MetaboAnalyst 4.0 to explore the metabolic reprogramming pathways involved in PSS. Logistic regression and receiver-operating characteristic (ROC) analyses were employed to evaluate the diagnostic capability of selected metabolites. Comparative analysis revealed a clear separation between PSS and control groups. Fourteen novel differentiating metabolites from AH samples obtained from patients with PSS were highlighted. Pathway analysis identified 11 carbohydrate, amino acid metabolism and energy metabolism pathways as the major disturbed pathways associated with PSS. The abnormal lysine degradation metabolism, valine-leucine-isoleucine biosynthesis, and citrate circle were considered to weigh the most in the development of PSS. The ROC analysis implied that the combination of glycine and homogentisic acid could serve as potential biomarkers for the discrimination of control and PSS groups. In conclusion, these results revealed for the first time the identity of important metabolites and pathways contributing to the development/progression of PSS, enabled the better understanding of the mechanism of PSS, and might lead to the development of metabolic biomarkers and novel therapeutic strategies to restrict the development/progression of PSS.

Project description:(1) Rho-associated coiled-coil protein kinase (ROCK) signaling cascade impacts a wide array of cellular events. For cellular therapeutics, scalable expansion of primary human corneal endothelial cells (CECs) is crucial, and the inhibition of ROCK signaling using a well characterized ROCK inhibitor (ROCKi) Y-27632 had been shown to enhance overall endothelial cell yield. (2) In this study, we compared several classes of ROCK inhibitors to both ROCK-I and ROCK-II, using in silico binding simulation. We then evaluated nine ROCK inhibitors for their effects on primary CECs, before narrowing it down to the two most efficacious compounds-AR-13324 (Netarsudil) and its active metabolite, AR-13503-and assessed their impact on cellular proliferation in vitro. Finally, we evaluated the use of AR-13324 on the regenerative capacity of donor cornea with an ex vivo corneal wound closure model. Donor-matched control groups supplemented with Y-27632 were used for comparative analyses. (3) Our in silico simulation revealed that most of the compounds had stronger binding strength than Y-27632. Most of the nine ROCK inhibitors assessed worked within the concentrations of between 100 nM to 30 µM, with comparable adherence to that of Y-27632. Of note, both AR-13324 and AR-13503 showed better cellular adherence when compared to Y-27632. Similarly, the proliferation rates of CECs exposed to AR-13324 were comparable to those of Y-27632. Interestingly, CECs expanded in a medium supplemented with AR-13503 were significantly more proliferative in (i) untreated vs. AR-13503 (1 μM; * p < 0.05); (ii) untreated vs. AR-13503 (10 μM; *** p < 0.001); (iii) Y-27632 vs. AR-13503 (10 μM; ** p < 0.005); (iv) AR-13324 (1 μM) vs. AR-13503 (10 μM; ** p < 0.005); and (v) AR-13324 (0.1 μM) vs. AR-13503 (10 μM; * p < 0.05). Lastly, an ex vivo corneal wound healing study showed a comparable wound healing rate for the final healed area in corneas exposed to Y-27632 or AR-13324. (4) In conclusion, we were able to demonstrate that various classes of ROCKi compounds other than Y-27632 were able to exert positive effects on primary CECs, and systematic donor-match controlled comparisons revealed that the FDA-approved ROCK inhibitor, AR-13324, is a potential candidate for cellular therapeutics or as an adjunct drug in regenerative treatment for corneal endothelial diseases in humans.

Dataset Information

Effects of ROCK Inhibitors on Apoptosis of Corneal Endothelial Cells in CMV-Positive Posner-Schlossman Syndrome Patients.

Publications

Effects of ROCK Inhibitors on Apoptosis of Corneal Endothelial Cells in CMV-Positive Posner-Schlossman Syndrome Patients.

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